A novel metric-based approach of scoring early host immune response from oro-nasopharyngeal swabs predicts COVID-19 outcome.

Unpredictable fatal outcome of COVID-19 is attributed to dysregulated inflammation. Impaired early adaptive immune response leads to late-stage inflammatory outcome. The purpose of this study was to develop biomarkers for early detection of host immune impairment at first diagnosis from leftover RNA samples, which may in turn identify high risk patients. Leftover RNA samples of COVID-19 patients at first diagnosis were stored. Following prospective follow-up, the samples were shorted and categorized into outcome groups. Impaired adaptive T cell response (severity score) and Impaired IL-10 response (undetectable IL-10 in the presence of high expression of a representative interferon response gene) were determined by RT-PCR based assay. We demonstrate that a T cell response based 'severity score' comprising rational combination of Ct values of a target genes' signature can predict high risk noncomorbid potentially critical COVID-19 patients with a sensitivity of 91% (95% CI 58.7-99.8) and specificity of 92.6% (95% CI 75.7-99) (AUC:0.88). Although inclusion of comorbid patients reduced sensitivity to 77% (95% CI 54.6-92.2), the specificity was still 94% (95% CI 79.8-99.3) (AUC:0.82). The same for 'impaired IL-10 response' were little lower to predict high risk noncomorbid patients 64.2% (95% CI 35.1-87.2) and 82% (95% CI 65.5-93.2) respectively. Inclusion of comorbid patients drastically reduce sensitivity and specificity51.6% (95% CI 33.1-69.8) and 80.5% (95% CI 64.0-91.8) respectively. As best of our knowledge this is the first demonstration of a metric-based approach showing the 'severity score' as an indicator of early adoptive immune response, could be used as predictor of severe COVID-19 outcome at the time of first diagnosis using the same leftover swab RNA. The work flow could reduce expenditure and reporting time of the prognostic test for an earliest clinical decision ensuring possibility of early rational management.

Effectiveness of sample pooling strategies for diagnosis of SARS-CoV-2: Specimen pooling vs. RNA elutes pooling.

PURPOSE: The pandemic of SARS-CoV-2 or COVID-19 has hugely created an economic imbalance worldwide. With the exponential increase in the number of cases and to keep in check on the community transmission, there is high demand and acute shortage of diagnostic kits. The pooled-sample strategy turns out to be the promising strategy intended to determine the optimal testing for specimens with limited resources and without losing the test sensitivity and specificity. The study was performed with standard molecular biology graded lab equipment, FDA-approved COVID-19 RNA extraction, and SARS-CoV-2 tests kits. MATERIALS AND METHODS: The study aims to comparatively analyze the pooling strategy of the naso-oropharyngeal specimen sample and RNA extracted from the same patient samples in the pool of 3,5, and 8 with no significant loss in test usability. Another primary focus of the study was detection of low or borderline SARS-CoV-2 positives in the pooling strategy. A total of 300 samples (240 positives and 60 negatives) were tested for 3, 5, and 8 pools of specimen samples and RNA elutes. RESULTS: The comparative analysis determined the sensitivity for three and five pool strategy to be above 98% and eight pool strategy to be 100%. CONCLUSION: The RNA elutes pooling strategy concordance rate is better than that of specimen pooling with 100% specificity. Thus, in the substantial crisis of resources with the global pandemic, pooling approaches for SARS-CoV-2 can be practical in a low prevalence rate of 5%.

Constitutive expression and discovery of antimicrobial peptides in Zygogramma bicolorata (Coleoptera: Chrysomelidae).

The expression, identification, and discovery of less toxic antimicrobial peptides (AMPs) are significant in managing infectious pathogens. AMPs triggered in response to the immune system have evolved to defend against pathogens and wounding. The protein composition of Zygogramma bicolorata hemolymph is of diagnostic importance as the open circulatory systems of the insects involve signaling through hemolymph. They have conserved many ancestral vertebrate genes that may help better understand the evolution of innate immunity. The present work describes the isolation, purification, identification, and bioinformatics analysis of AMPs from the immunized hemolymph of Z. bicolorata. Thirty-nine peptides were isolated from reverse-phase high-performance liquid chromatography and sequenced via mass spectrometry analysis. The immunization process recorded a threefold higher protein concentration in immunized hemolymph when compared with nonimmunized one. For the first time, the proteomic study on Z. bicolorata hemolymph unveils the three novel proteins in the family Chrysomelidae with no homology in the database, indicating its novelty and the expression of the rest of 36 well-known proteins, including heat-shock, immune, structural, signaling proteins, and others speak for its method validity. Combining the expression of novel AMPs, detoxifying enzymes, hemolytic, and cytotoxic assays, and this work can elucidate new pathways to immune response mechanisms. Its molecular basis also holds the potential applicability in the future drug development process against pathogenic fungi such as Aspergillus niger and Candida albicans.

VEGF Promoter Region 18-bp Insertion-Deletion Polymorphism in Sickle Cell Disease Patients with Microalbuminuria: A Pilot Study.

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent inducer of micro vascular permeability thus leading to nephropathy. Insertion/deletion (I/D) polymorphism of 18 bp at - 2549 position in VEGF gene causes increased transcription leading to increased production of VEGF. Thus, we aimed to associate I/D polymorphism of the 18 bp fragment at - 2549 position of the promoter region of VEGF gene with sickle cell nephropathy (SCN). METHODS: This observational analytical case control study included 30 subjects each of SCN, sickle cell disease (SCD) without nephropathy and the control group. The subjects were assessed for various hematological and biochemical parameters. Further, 18 bp I/D polymorphism of VEGF gene in all three study groups was assessed by polymerase chain reaction followed by electrophoresis and compared. RESULT: Though increased frequency of both DD genotype and D allele was found in SCN compared to SCD and control, only frequency of D allele was found to be significantly higher (p = 0.04). D allele posed marginal risk of microalbuminuria in SCD subjects compared to controls (OR = 2.11) as well as to SCD without MA subjects (OR = 1.84). CONCLUSION: D allele in I/D polymorphism in the promoter region of VEGF gene may be associated with marginal increase in risk of susceptibility to sickle cell nephropathy.

Polymorphism of CYP1A1 gene variants rs4646903 and rs1048943 relation to the incidence of cervical cancer in Chhattisgarh.

Cytochrome P450 CYP1A1 is a phase 1 xenobiotic metabolizing enzyme involved in the metabolism of toxins, endogenous hormones and pharmaceutical drugs. It is therefore possible that polymorphism of CYP1A1 gene producing functional changes in the enzyme may be susceptible factors in cervical carcinogenesis. This study was aimed to look association of CYP1A1m1 (T>C) and m2 (A>G) gene polymorphisms in Chhattisgarh population. In this case-control study, we analyzed leukocyte DNA from a total of 200 subjects form Chhattisgarh (100 cases and 100 controls). All subjects were genotyped for CYP1A1m1 (T>C) and m2 (A>G) using PCR-RFLP with statistical analysis by using SPSS version 16.0 and VassarStats (online). Among the two gene variants rs4646903 (T>C) and rs1048943 (A>G), individuals with AG and GG genotypes of CYP1A1m2 polymorphism have significantly higher and increased risk of cervical cancer (OR=2.0, 95%CI=1.04-3.84, p=0.035; OR=62.9, 95%CI=3.72-1063.83, p=0.004 respectively) and the association of CYP1A1m1 polymorphism did not show any significant relationship with cervical cancer patients (p=0.23). The 'G' allele showed strong association with the disease (p<0.0001). Thus, CYP1A1m2 polymorphism showed an increased risk in the population leading to cervical cancer. Our study suggested that the presence of 'C' allele of rs4646903 (T>C) showed no risk and 'G' allele of rs1048943 (A>G) might be a leading allele to cause increased cervical cancer susceptibility due to significant association of CYP1A1m2 gene polymorphism.